Project description:Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites and cellular energy status are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Stoichiometric analysis revealed that de novo resveratrol production from glucose requires a net input of 2 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates, a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g., 27% at 0.03 h-1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in the chemostats. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g., ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways.

Project description:Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites and cellular energy status are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Stoichiometric analysis revealed that de novo resveratrol production from glucose requires a net input of 2 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates, a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g., 27% at 0.03 h-1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in the chemostats. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g., ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways. The goal of the present study is to investigate the impact of specific growth rate on biomass-specific productivity, product yield, by-product formation and host strain physiology of an S. cerevisiae strain that was previously engineered for de novo production of resveratrol from glucose. To this end, (by)product formation, physiology and transcriptome were analysed in steady-state, glucose-limited chemostat cultures grown at different dilution rates.

Project description:The transcriptome profiles for wild-type (plasmid-free) and recombinant (plasmid-bearing) Escherichia coli during well-controlled synchronized high-cell-density fed-batch cultures were analyzed by DNA microarrays. It was observed that the growth phase significantly affected the transcriptome profiles, and the transcriptome profiles were significantly different for the recombinant and wild-type cultures. The response of the wild-type and recombinant cultures to an isopropyl-1-thio-β-D-galactopyranoside- (IPTG-) addition was examined, where IPTG induced recombinant protein production in the plasmid-bearing cultures. The IPTG-addition significantly altered the transcriptome response of the wild-type cultures entering the stationary phase. The IPTG-induced recombinant protein production resulted in a significant down-regulation of many energy synthesis genes (atp, nuo, cyo), as well as nearly all transcription- and translation-related genes (rpo, rpl, rpm, rps, rrf, rrl, rrs). Numerous phage (psp, hfl) and transposon-related genes (tra, ins) were significantly regulated in the recombinant cultures due to the IPTG-induction. These results indicate that the signaling mechanism, associated with the recombinant protein production, may induce a metabolic burden in the form of a phage defense mechanism. Taken together, these results indicated that recombinant protein production initiated a cascade of transcriptome responses that down-regulated the very genes needed to sustain productivity. In this study, the transcriptome profiles for recombinant and wild-type E. coli cultures were compared. The recombinant and wild-type cultures were monitored during the exponential growth phase and as the cultures entered the stationary phase. Well-controlled fed-batch fermenters were used to synchronize the high-cell density cultures. E. coli MG1655 [pPROEx-CAT] and E. coli MG1655 (plasmid-free) were exposed to IPTG in the exponential phase, where chloramphenicol acetyltransferase (CAT) expression was induced in the plasmid-bearing cultures. Control cultures, not exposed to IPTG, were also examined. The gene expression profiles were determined using DNA microarrays. Recombinant protein production was shown to result in a metabolic burden due to significant down-regulation of the transcription, translation, and energy synthesis genes. This was the first comprehensive study that has examined the transcriptome of wild-type and recombinant cultures under the same set of conditions. This data indicated that one cannot merely extrapolate the behavior of recombinant cultures from wild-type culture data.

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Analyzing the metabolic stress response of recombinant Escherichia coli cultures expressing human interferon beta in high cell density fed batch cultures using time course transcriptomic data

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